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Replacement of pre-T cell receptor signaling functions by the CD4 coreceptor.

Norment AM, Forbush KA, Nguyen N, Malissen M, Perlmutter RM - J. Exp. Med. (1997)

Bottom Line: However, the biochemical mechanisms governing p56lck activation remain poorly understood.In more mature thymocytes, p56lck is associated with the cytoplasmic domain of the TCR coreceptors CD4 and CD8, and cross-linking of CD4 leads to p56lck activation.We show that this process is dependent upon the ability of the CD4 transgene to bind Lck and on the expression of MHC class II molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle 98195, USA.

ABSTRACT
An important checkpoint in early thymocyte development ensures that only thymocytes with an in-frame T cell receptor for antigen beta (TCR-beta) gene rearrangement will continue to mature. Proper assembly of the TCR-beta chain into the pre-TCR complex delivers signals through the src-family protein tyrosine kinase p56lck that stimulate thymocyte proliferation and differentiation to the CD4+CD8+ stage. However, the biochemical mechanisms governing p56lck activation remain poorly understood. In more mature thymocytes, p56lck is associated with the cytoplasmic domain of the TCR coreceptors CD4 and CD8, and cross-linking of CD4 leads to p56lck activation. To study the effect of synchronously inducing p56lck activation in immature CD4-CD8- thymocytes, we generated mice expressing a CD4 transgene in Rag2-/- thymocytes. Remarkably, without further experimental manipulation, the CD4 transgene drives maturation of Rag2-/- thymocytes in vivo. We show that this process is dependent upon the ability of the CD4 transgene to bind Lck and on the expression of MHC class II molecules. Together these results indicate that binding of MHC class II molecules to CD4 can deliver a biologically relevant, Lck-dependent activation signal to thymocytes in the absence of the TCR-alpha or -beta chain.

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Flow cytometric analysis of thymocytes from CD4Tg+ Rag2−/−  mice mated onto an MHC class II−/− background. (A) Fluorescence staining profiles of CD4 and CD8 (top panels) or the MHC class II molecule I-Ab (bottom panels)  of thymocytes from a CD4 transgene negative MHC class II wild-type control  (Rag2−/− MHC II+/+) and CD4 transgene positive littermates that are wild-type  (CD4Tg+ Rag2−/− MHC II+/+), heterozygous (CD4Tg+ Rag2−/− MHC II+/−) or   (CD4Tg+ Rag2−/− MHC II−/−) for MHC class II expression. The percentage  of cells in each population is indicated. Unlike thymi from CD4Tg+ Rag2−/−  mice that were first analyzed (Fig. 1), thymi from the crosses of CD4Tg+ Rag2−/−  mice with MHC class II−/− mice did not exhibit an increase in cell number relative to CD4 transgene negative littermates. This was observed even in CD4Tg+  Rag2−/− MHC class II+/+ progeny of MHC class II heterozygote crosses. This is  likely due to nonspecific changes in genetic background. (B) Scatter graph of the  percentage of CD4+CD8+ thymocytes from the progeny of CD4Tg+ Rag2−/− ×  MHC class II−/− matings. Each circle shows a data point from an individual  mouse, with the mean represented by a bold plus sign (+). The number of mice  analyzed for each type is indicated across the top of the graph. CD4 transgene  negative littermate controls (LMC) included MHC class II , heterozygous  and wild-type mice.
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Figure 3: Flow cytometric analysis of thymocytes from CD4Tg+ Rag2−/− mice mated onto an MHC class II−/− background. (A) Fluorescence staining profiles of CD4 and CD8 (top panels) or the MHC class II molecule I-Ab (bottom panels) of thymocytes from a CD4 transgene negative MHC class II wild-type control (Rag2−/− MHC II+/+) and CD4 transgene positive littermates that are wild-type (CD4Tg+ Rag2−/− MHC II+/+), heterozygous (CD4Tg+ Rag2−/− MHC II+/−) or (CD4Tg+ Rag2−/− MHC II−/−) for MHC class II expression. The percentage of cells in each population is indicated. Unlike thymi from CD4Tg+ Rag2−/− mice that were first analyzed (Fig. 1), thymi from the crosses of CD4Tg+ Rag2−/− mice with MHC class II−/− mice did not exhibit an increase in cell number relative to CD4 transgene negative littermates. This was observed even in CD4Tg+ Rag2−/− MHC class II+/+ progeny of MHC class II heterozygote crosses. This is likely due to nonspecific changes in genetic background. (B) Scatter graph of the percentage of CD4+CD8+ thymocytes from the progeny of CD4Tg+ Rag2−/− × MHC class II−/− matings. Each circle shows a data point from an individual mouse, with the mean represented by a bold plus sign (+). The number of mice analyzed for each type is indicated across the top of the graph. CD4 transgene negative littermate controls (LMC) included MHC class II , heterozygous and wild-type mice.

Mentions: We next asked whether the presence of CD4 in immature thymocytes was by itself sufficient to stimulate maturation, or whether interaction with an MHC class II ligand was required. The extracellular domain of CD4 binds directly to the MHC class II β2 domain (29, 30), and it is well established that CD4 coreceptor function requires MHC class II binding in mature T cells (31). To determine whether CD4 transgene-driven thymocyte maturation in Rag2−/− mice is dependent upon MHC class II molecules, CD4Tg+ Rag2−/− mice were crossed with MHC class II−/− mice (generated through targeted disruption of the I-Aβ locus [27]). Rag2−/−, CD4Tg, and MHC class II−/− mice share the H-2bhaplotype, and therefore lack surface expression of MHC class II I-E proteins due to a pre-existing mutation in the Eα gene promoter (32). As shown in Fig. 3 A, in the absence of MHC class II expression (right panel), the CD4 transgene no longer stimulates the acquisition of CD8 expression in Rag2−/− thymocytes. Indeed, in CD4Tg mice made simultaneously for Rag2 and heterozygous for the disrupted I-Aβ allele, the representation of DP cells typically reached only 40% of that observed in those bearing two wild-type I-Aβ alleles (Fig. 3 A, middle panels and B). Hence the actual dose of MHC class II molecules available to bind CD4 appears to regulate propagation of a differentiative signal.


Replacement of pre-T cell receptor signaling functions by the CD4 coreceptor.

Norment AM, Forbush KA, Nguyen N, Malissen M, Perlmutter RM - J. Exp. Med. (1997)

Flow cytometric analysis of thymocytes from CD4Tg+ Rag2−/−  mice mated onto an MHC class II−/− background. (A) Fluorescence staining profiles of CD4 and CD8 (top panels) or the MHC class II molecule I-Ab (bottom panels)  of thymocytes from a CD4 transgene negative MHC class II wild-type control  (Rag2−/− MHC II+/+) and CD4 transgene positive littermates that are wild-type  (CD4Tg+ Rag2−/− MHC II+/+), heterozygous (CD4Tg+ Rag2−/− MHC II+/−) or   (CD4Tg+ Rag2−/− MHC II−/−) for MHC class II expression. The percentage  of cells in each population is indicated. Unlike thymi from CD4Tg+ Rag2−/−  mice that were first analyzed (Fig. 1), thymi from the crosses of CD4Tg+ Rag2−/−  mice with MHC class II−/− mice did not exhibit an increase in cell number relative to CD4 transgene negative littermates. This was observed even in CD4Tg+  Rag2−/− MHC class II+/+ progeny of MHC class II heterozygote crosses. This is  likely due to nonspecific changes in genetic background. (B) Scatter graph of the  percentage of CD4+CD8+ thymocytes from the progeny of CD4Tg+ Rag2−/− ×  MHC class II−/− matings. Each circle shows a data point from an individual  mouse, with the mean represented by a bold plus sign (+). The number of mice  analyzed for each type is indicated across the top of the graph. CD4 transgene  negative littermate controls (LMC) included MHC class II , heterozygous  and wild-type mice.
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Figure 3: Flow cytometric analysis of thymocytes from CD4Tg+ Rag2−/− mice mated onto an MHC class II−/− background. (A) Fluorescence staining profiles of CD4 and CD8 (top panels) or the MHC class II molecule I-Ab (bottom panels) of thymocytes from a CD4 transgene negative MHC class II wild-type control (Rag2−/− MHC II+/+) and CD4 transgene positive littermates that are wild-type (CD4Tg+ Rag2−/− MHC II+/+), heterozygous (CD4Tg+ Rag2−/− MHC II+/−) or (CD4Tg+ Rag2−/− MHC II−/−) for MHC class II expression. The percentage of cells in each population is indicated. Unlike thymi from CD4Tg+ Rag2−/− mice that were first analyzed (Fig. 1), thymi from the crosses of CD4Tg+ Rag2−/− mice with MHC class II−/− mice did not exhibit an increase in cell number relative to CD4 transgene negative littermates. This was observed even in CD4Tg+ Rag2−/− MHC class II+/+ progeny of MHC class II heterozygote crosses. This is likely due to nonspecific changes in genetic background. (B) Scatter graph of the percentage of CD4+CD8+ thymocytes from the progeny of CD4Tg+ Rag2−/− × MHC class II−/− matings. Each circle shows a data point from an individual mouse, with the mean represented by a bold plus sign (+). The number of mice analyzed for each type is indicated across the top of the graph. CD4 transgene negative littermate controls (LMC) included MHC class II , heterozygous and wild-type mice.
Mentions: We next asked whether the presence of CD4 in immature thymocytes was by itself sufficient to stimulate maturation, or whether interaction with an MHC class II ligand was required. The extracellular domain of CD4 binds directly to the MHC class II β2 domain (29, 30), and it is well established that CD4 coreceptor function requires MHC class II binding in mature T cells (31). To determine whether CD4 transgene-driven thymocyte maturation in Rag2−/− mice is dependent upon MHC class II molecules, CD4Tg+ Rag2−/− mice were crossed with MHC class II−/− mice (generated through targeted disruption of the I-Aβ locus [27]). Rag2−/−, CD4Tg, and MHC class II−/− mice share the H-2bhaplotype, and therefore lack surface expression of MHC class II I-E proteins due to a pre-existing mutation in the Eα gene promoter (32). As shown in Fig. 3 A, in the absence of MHC class II expression (right panel), the CD4 transgene no longer stimulates the acquisition of CD8 expression in Rag2−/− thymocytes. Indeed, in CD4Tg mice made simultaneously for Rag2 and heterozygous for the disrupted I-Aβ allele, the representation of DP cells typically reached only 40% of that observed in those bearing two wild-type I-Aβ alleles (Fig. 3 A, middle panels and B). Hence the actual dose of MHC class II molecules available to bind CD4 appears to regulate propagation of a differentiative signal.

Bottom Line: However, the biochemical mechanisms governing p56lck activation remain poorly understood.In more mature thymocytes, p56lck is associated with the cytoplasmic domain of the TCR coreceptors CD4 and CD8, and cross-linking of CD4 leads to p56lck activation.We show that this process is dependent upon the ability of the CD4 transgene to bind Lck and on the expression of MHC class II molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle 98195, USA.

ABSTRACT
An important checkpoint in early thymocyte development ensures that only thymocytes with an in-frame T cell receptor for antigen beta (TCR-beta) gene rearrangement will continue to mature. Proper assembly of the TCR-beta chain into the pre-TCR complex delivers signals through the src-family protein tyrosine kinase p56lck that stimulate thymocyte proliferation and differentiation to the CD4+CD8+ stage. However, the biochemical mechanisms governing p56lck activation remain poorly understood. In more mature thymocytes, p56lck is associated with the cytoplasmic domain of the TCR coreceptors CD4 and CD8, and cross-linking of CD4 leads to p56lck activation. To study the effect of synchronously inducing p56lck activation in immature CD4-CD8- thymocytes, we generated mice expressing a CD4 transgene in Rag2-/- thymocytes. Remarkably, without further experimental manipulation, the CD4 transgene drives maturation of Rag2-/- thymocytes in vivo. We show that this process is dependent upon the ability of the CD4 transgene to bind Lck and on the expression of MHC class II molecules. Together these results indicate that binding of MHC class II molecules to CD4 can deliver a biologically relevant, Lck-dependent activation signal to thymocytes in the absence of the TCR-alpha or -beta chain.

Show MeSH
Related in: MedlinePlus